Alan M Lambowitz

US Patent:

7592161, Sep 22, 2009

Filed:

Oct 22, 2002

Appl. No.:

10/277643

Inventors:

Alan M. Lambowitz - Austin TX, US
Huatao Guo - Alhambra CA, US
Michael Karberg - Austin TX, US

Assignee:

The Ohio State University Research Foundation - Columbus OH
The University of Texas Board of Regents of the University of Texas System - Austin TX

International Classification:

C12P 19/34
C12P 12/06

US Classification:

435 911, 435 691, 435194, 536 232, 536 234, 536 241

Abstract:

The present invention provides a system and methods for analyzing the function of nucleotide integrases and modified group II introns. The system comprises a donor plasmid comprising a wild-type or modified group II intron, a recipient plasmid comprising a DNA recognition site and a promoterless reporter gene downstream of the DNA target site, and a host cell. The method comprises the steps of transforming a host cell with the donor and recipient plasmids, assaying for expression of the reporter gene, isolating plasmid DNA from the cotransformed cells, and analyzing the plasmid DNA to confirm that the group II intron has been inserted into the target sequence. The present invention also provides a method for simultaneously analyzing the activity of two or more modified nucleotide integrases. The present invention also relates to methods of preparing a library of donor plasmids containing a plurality of diverse modified group II intron DNA sequences.

US Patent:

20020086323, Jul 4, 2002

Filed:

Oct 22, 2001

Appl. No.:

10/008618

Inventors:

Alan Lambowitz - Austin TX, US
Steven Zimmerly - Calgary, CA
Huatao Guo - Austin TX, US
Georg Mohr - Austin TX, US
Clifford Beall - Columbus OH, US

International Classification:

C12Q001/68
C12P019/34

US Classification:

435/006000, 435/091200

Abstract:

Methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double-stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate are provided. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing an isolated nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on the top strand of the DNA substrate, and a group I-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate to permit the nucleotide integrase to cleave the top strand of the DNA substrate and to insert the group II intron RNA into the cleavage site. The method of cleaving both strands of a double-stranded DNA substrate comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on one strand of the substrate, and a group II-intron encoded protein that interacts with a first sequence element and a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate such that the nucleotide integrase cleaves both strands of the DNA substrate and inserts the group II intron RNA into the cleavage site of the one strand. The method for cleaving a single-stranded nucleic acid substrate comprises the steps of: providing a nucleotide integrase having two hybridizing sequences for hybridizing with two intron RNA-binding sequences on the single-stranded substrate, and a group II intron encoded protein; and reacting the nucleotide integrase with the single stranded nucleic acid substrate to allow the nucleotide integrase to cleave the substrate and to attach the group II intron RNA molecule thereto.

US Patent:

20090142838, Jun 4, 2009

Filed:

Jun 14, 2005

Appl. No.:

11/629441

Inventors:

Xiaoxia Cui - Saint Louis MO, US
Alan Lambowitz - Austin TX, US
Roland Saldanha - Austin TX, US

International Classification:

C12N 15/87
C12N 15/00

US Classification:

435455, 4353201

Abstract:

Provided herein are nucleic acid constructs and methods for producing or enhancing the production of group II intron RNP particles in eukaryotic cells. The present methods comprise introducing at least one nucleic acid construct comprising a nucleic acid encoding a modified or wild type group II intron RNA and a wild-type or modified group II intron-encoded protein into the eukaryotic cell, and maintaining the cell under conditions that allow for expression of the group II intron RNA and the group II intron-encoded protein in the cell. The nucleic acid encoding the group II intron RNA is operably linked to an RNA polymerase I, an RNA polymerase II, or an RNA polymerase III promoter, and the nucleic acid encoding the group II intron-encoded protein is operably linked to an RNA polymerase II promoter. In certain embodiments, a subcellular localization signal is attached to the group II intron-encoded protein.

US Patent:

20120009630, Jan 12, 2012

Filed:

Mar 4, 2010

Appl. No.:

13/254223

Inventors:

Alan M. Lambowitz - Austin TX, US
Sabine Mohr - Austin TX, US
Georg Mohr - Austin TX, US
Eman Ghanem - Austin TX, US

Assignee:

Board of Regents, The University of Texas System - Austin TX

International Classification:

C12P 19/34
C12N 15/63
C12N 9/12

US Classification:

435 9151, 435194, 4353201

Abstract:

Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

US Patent:

20140004569, Jan 2, 2014

Filed:

Feb 23, 2012

Appl. No.:

14/000513

Inventors:

Alan M. Lambowitz - Austin TX, US
Sabine Mohr - Austin TX, US
Travis B. White - Austin TX, US
Scott Kuersten - Fitchburg WI, US

Assignee:

Board of Regents, The University of Texas System - Austin TX

International Classification:

C12P 19/34

US Classification:

435 9121, 435 912

Abstract:

A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non-retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3′ end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

US Patent:

58044183, Sep 8, 1998

Filed:

Nov 19, 1996

Appl. No.:

8/752238

Inventors:

Alan Marc Lambowitz - Columbus OH
Georg Mohr - Columbus OH
Roland Saldanha - Columbus OH
Manabu Matsuura - Columbus OH

Assignee:

The Ohio State University Research Foundation - Columbus OH

International Classification:

C12P 2102
C12P 1934
C12N 912
C12N 1554

US Classification:

435 691

Abstract:

The present invention provides new, improved, and easily manipulable methods for making nucleotide integrases. In one embodiment, the nucleotide integrase is prepared by introducing a DNA molecule which comprises a group II intron DNA sequence into a host cell. The group II intron DNA sequence is then expressed in the host cell such that RNP particles having nucleotide integrase activity are formed in the cell. Such RNP particles comprise an exiced group II intron RNA encoded by the introduced DNA molecule and a group II intron-encoded protein encoded by the introduced DNA molecule. Thereafter, the nucleotide integrase is isolated from the cell. In another embodiment, the nucleotide integrase is prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as "exogenous RNA", with a group II intron-encoded protein. In another embodiment, the nucleotide integrase is prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as "exogenous RNA", with an RNA-protein complex which comprises a group II intron-encoded protein. Preferably, the exogenous RNA is prepared by in vitro transcription of a DNA molecule which comprises the group II intron sequence.

US Patent:

56984219, Dec 16, 1997

Filed:

Sep 12, 1995

Appl. No.:

8/526964

Inventors:

Alan M. Lambowitz - Columbus OH
Steven Zimmerly - Columbus OH
Jian Yang - Columbus OH
Huatao Guo - Columbus OH

Assignee:

The Ohio State Research Foundation - Columbus OH

International Classification:

C12P 1934
C12Q 168
C07H 2102
C07H 2104

US Classification:

435 911

Abstract:

The present invention provides new methods, employing a nucleotide integrase, for cleaving double-stranded and single stranded DNA substrates at specific sites and for attaching nucleic acid molecules to the cleaved DNA substrates. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA and to concomitantly attach a nucleic acid molecule to the cleaved strand. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach a nucleic acid molecule to one strand of the DNA substrate. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach an RNA molecule to one strand of the substrate and for attaching a cDNA to the other strand of the substrate. Another method cleaves single stranded DNA with the concomitant insertion of a nucleic acid molecule at the cleavage point. The nucleotide integrase comprises an RNP particle which comprises a group II intron RNA bound to a group II intron encoded protein.

US Patent:

58696342, Feb 9, 1999

Filed:

Oct 7, 1997

Appl. No.:

8/946617

Inventors:

Alan M. Lambowitz - Columbus OH
Steven Zimmerly - Columbus OH
Jian Yang - Columbus OH
Huatao Guo - Columbus OH

Assignee:

The Ohio State Research Foundation - Columbus OH

International Classification:

C07H 2102
C07K 1400
C12P 1934
C12Q 168

US Classification:

536 231

Abstract:

The present invention provides new methods, employing a nucleotide integrase, for cleaving double-stranded and single stranded DNA substrates at specific sites and for attaching nucleic acid molecules to the cleaved DNA substrates. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA and to concomitantly attach a nucleic acid molecule to the cleaved strand. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach a nucleic acid molecule to one strand of the DNA substrate. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach an RNA molecule to one strand of the substrate and for attaching a cDNA to the other strand of the substrate. Another method cleaves single stranded DNA with the concomitant insertion of a nucleic acid molecule at the cleavage point. The nucleotide integrase comprises an RNP particle which comprises a group II intron RNA bound to a group II intron encoded protein.